U.S. patent application number 14/906806 was filed with the patent office on 2016-06-09 for polysaccharide hydrogels for injection with tunable properties.
The applicant listed for this patent is ALBERT-LUDWIGS-UNIVERSITAT FREIBURG, UNIVERSITATSSPITAL BASEL. Invention is credited to Andrea BANFI, Aurelien FORGET, Roberto GIANNI-BARRERA, Prasad V. SHASTRI.
Application Number | 20160158410 14/906806 |
Document ID | / |
Family ID | 48803465 |
Filed Date | 2016-06-09 |
United States Patent
Application |
20160158410 |
Kind Code |
A1 |
SHASTRI; Prasad V. ; et
al. |
June 9, 2016 |
POLYSACCHARIDE HYDROGELS FOR INJECTION WITH TUNABLE PROPERTIES
Abstract
Injectable hydrogels comprising polysaccharides based on
disaccharides the backbones of which form an .alpha.-helix
structure and in which in at least 10% of the disaccharide units
the primary hydroxyl groups are oxidized.
Inventors: |
SHASTRI; Prasad V.;
(Freiburg, DE) ; BANFI; Andrea; (Basel, CH)
; FORGET; Aurelien; (Freiburg, DE) ;
GIANNI-BARRERA; Roberto; (Maennedorf, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALBERT-LUDWIGS-UNIVERSITAT FREIBURG
UNIVERSITATSSPITAL BASEL |
Freiburg
Basel |
|
DE
CH |
|
|
Family ID: |
48803465 |
Appl. No.: |
14/906806 |
Filed: |
July 21, 2014 |
PCT Filed: |
July 21, 2014 |
PCT NO: |
PCT/EP2014/065649 |
371 Date: |
January 21, 2016 |
Current U.S.
Class: |
514/8.1 ;
514/773; 514/777; 514/8.2; 514/8.5; 514/8.9; 514/9.1 |
Current CPC
Class: |
A61K 38/18 20130101;
A61L 27/52 20130101; A61L 27/20 20130101; A61L 2400/06 20130101;
A61K 9/06 20130101; A61K 47/36 20130101; A61K 9/0019 20130101; A61L
2300/414 20130101; A61L 27/22 20130101; A61L 27/54 20130101; A61L
27/3633 20130101 |
International
Class: |
A61L 27/20 20060101
A61L027/20; A61L 27/36 20060101 A61L027/36; A61L 27/54 20060101
A61L027/54; A61L 27/52 20060101 A61L027/52; A61L 27/22 20060101
A61L027/22 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 22, 2013 |
EP |
13177505.8 |
Claims
1. Injectable hydrogels comprising polysaccharides based on
disaccharides the backbones of which form an a-helix structure and
in which in at least 10% of the disaccharide units the primary
hydroxyl groups are oxidized.
2. Injectable hydrogels in accordance with claim 1 wherein in at
least 20% of the disaccharide units the primary hydroxyl groups are
oxidized.
3. Injectable hydrogels in accordance with claim 1 or 2 wherein in
at least 35% of the disaccharide units the primary hydroxyl groups
are oxidized.
4. Injectable hydrogels in accordance with claim 1 or 2 wherein in
50 to 95% of the disaccharide units the primary hydroxyl groups are
oxidized.
5. Injectable hydrogels in accordance with claim 1 or 2 wherein in
55 to 93% of the disaccharide units the primary hydroxyl groups are
oxidized.
6. Injectable hydrogels in accordance with any of claims 1 to 5
wherein the polysaccharide is selected from the group consisting of
agarose and .kappa.-carrageenan.
7. Injectable hydrogels in accordance with any of claims 1 to 6
wherein the polysaccharide is modified with cell adhesion motifs
such as the integrin binding sequence arginine-glycine-aspartic
acid (RGD), or with peptide sequences.
8. Injectable hydrogels in accordance with claim 7 wherein the
peptide sequence is selected from YIGSR, IKVAV, MNYYSNS or
PHSRN.
9. Injectable hydrogels in accordance with any of claims 1 to 8
comprising soluble signals.
10. Injectable hydrogels in accordance with claim 9 wherein the
soluble signal is selected from vascular endothelial growth factor
(VEGF), phorbol 12 myristate acetate (PMA), fibroblast growth
factors (FGF), insulin growth factors (IGF), transforming growth
factor beta-1(TGF-.beta.) or platelet derived growth factor
(PDGF).
11. Injectable hydrogels in accordance with any of claims 1 to 10
comprising components of the extracellular matrix (ECM).
12. Injectable hydrogels in accordance with claim 11 wherein the
components of the extracellular matrix are selected from basement
membrane proteins (BMP).
13. Injectable hydrogels in accordance with claim 12 wherein the
basement membrane protein is selected from collagen type 4 (Col4),
laminins (LAM) or entactin (also known as nidogen) or mixtures
thereof.
14. Use of the injectable hydrogels in accordance with any of
claims 1 to 13 in therapeutic angiogenesis.
15. A method for reducing the shear modulus G' of hydrogels of
polysaccharides based on disaccharides the backbones of which form
an .alpha.-helical structure wherein the hydrogels are subjected to
oxidation of the primary hydroxyl groups of the disaccharide
units.
16. A process for inducing new vasculature in tissue comprising the
use of injectable hydrogels in accordance with any of claims 1 to
13.
Description
[0001] The present invention relates to polysaccharide hydrogels
with tunable properties like stiffness and provasculogenic
properties.
[0002] The organization of cells into tissue-like structures
involves a complex interplay between soluble signals and those
originating from the extracellular matrix (ECM).
[0003] In recent years several studies have shown that cells can
respond to physical cues (substrate stiffness and nanoroughness) in
a very well-defined manner, and this may constitute a new form of
signaling (1-3). Mechanobiology--the interplay between biological
and physical signals in establishing cell function--constitutes a
new avenue for deciphering the signaling environment during tissue
morphogenesis. In this regard, there is a need to develop systems
that can enable the investigation and translation of mechanobiology
paradigms into regenerative medicine solutions in vivo (4-6).
[0004] Such a system should meet the following criteria: offer
precise tailoring of the mechanical environment in vivo, be
cytocompatible, enable predictable evolution of cellular function,
and exhibit human biocompatibility.
[0005] Hydrogels, by virtue of their ability to mimic several
aspects of physiological environments such as hydration state and
interconnected pore architecture, have been explored extensively in
this context as mimics of extracellular matrices (ECM (4)) and in
the de novo development of tissue (7-9).
[0006] Hydrogels can be formed from either synthetic or natural
water-soluble polymers, and the transformation of a polymer network
into a gel requires the introduction of cross-links (net points)
between polymer chains. Hydrogels of polyethylene glycol (PEG) and
hyaluronic acid (HA), an ECM component, constitute the most
prominent class of hydrogels for regenerative medicine
applications, and they are formed through radical
photopolymerization (10), Michael addition (vinyl sulfone) (11),
click chemistry (thiolene) (12)] or enzymatic (trans-glutaminase)
cross-linking (13). In addition to HA, other polysaccharides such
as alginate (14), which undergoes calcium-induced gelation (15,
16), and chitin (17) have also been explored. More recently,
self-assembled peptides have emerged as yet another class of
biologically derived hydrogels (18, 19).
[0007] To use hydrogels as instructive materials in the context of
mechanobiology, precise control over the mechanics and biology
within the hydrogel is desirable.
[0008] In a chemically cross-linked system varying the modulus
necessitates changing the polymer concentration and/or polymer
chain length. Additionally, implementation of chemical
cross-linking in vivo can be challenging as it requires initiators
and chemistries, which can also react with ECM components and
proteins, and when not consumed can lead to toxicity.
[0009] Agarose, a polysaccharide extracted from marine red algae
composed of D-galactose-3,6-anhydro-L-galactopyranose repeat units,
has received considerable attention in regenerative medicine in
recent years due to its cytocompatibility, tissue compatibility in
humans (20, 21), and ability to induce, in vivo, the de novo
formation of hyaline-like cartilage (8) and is currently undergoing
phase-3 clinical trials in humans as a carrier for chondrocytes
(22). Unlike PEG, HA, and alginate, agarose forms a hydrogel
through physical cross-linking (23), which in comparison with
chemical and ionic cross-linking offers several advantages
including the absence of reactive chemistry and ease of
implementation.
[0010] Vascularization is an important biological process that is
necessary for the development, repair and sustenance of tissue in
mammals. Vascularization is the formation of new blood vessels from
existing blood vessels through sprouting (angiogenesis) or the de
novo organization of endothelial progenitor cells into vascular
structures (vasculogenesis).
[0011] The constriction, or damage or loss of vasculature can lead
to irreversible damage to tissue and is the cause of many
pathologies and clinical conditions. For example, the constriction
of coronary arteries (vessels that supply oxygenated blood to the
heart muscle), which results in the formation of local ischemia
(loss of blood supply) will lead to damage to the heart tissue
resulting in a myocardial infarct.
[0012] Diabetes mellitus, a systemic disorder can lead to
peripheral vascular disease resulting in loss of function in the
extremities such as limbs due to ischemia and associated avascular
necrosis. Constriction or damage to vasculature in the limbs can
promote muscle degeneration.
[0013] In all of the aforementioned pathologies, the induction of
new blood vessels (angiogenesis or vasculogenesis) is considered to
be an important step in stabilizing or reversing the negative
effects of ischemia.
[0014] The deliberate induction of new vasculature in a tissue
through an external intervention is called Therapeutic Angiogenesis
(TA). The goal of therapeutic angiogenesis is to stimulate the
creation of new blood vessels in ischemic organs, tissues or parts
with the goal to increase the level of oxygen-rich blood reaching
these areas
[0015] Formation of new blood vessels can be triggered by the local
administration of proteins such as vascular endothelial growth
factor (VEGF), fibroblast growth factor (FGF), genetic material
(plasmid or viral vectors) that encode for VEGF or FGF and/or
endothelial progenitor cells (EPCs) with or without association
with an injectable carrier. These efforts have largely resulted in
the induction of vasculature that lacks appropriate physiological
structural and functional traits and tends to regress over a period
of time.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1A shows the circular dichroism (CD) spectrum of a
0.15% wt/vol solution of natural agarose (NA) and 93% carboxylated
agarose (93-CA) obtained below the gelation temperature.
[0017] FIG. 1B shows the plot of the ellipticity at 203 nm as a
function of the degree of carboxylation;
[0018] FIG. 1C shows a Ramachandran plot for natural agarose (NA)
and 93% carboxylated agarose (CA);
[0019] FIG. 2A shows the tapping mode atomic force microscopy (AFM)
of single molecule height (main plot) and phase (Inset) for natural
agarose and carboxylated agarose with various degrees of
carboxylation;
[0020] FIG. 2B shows the Environmental Scanning Electron Microscopy
(ESEM) of freeze dried 2% wt/vol hydrogel of natural agarose and
carboxylated agarose with various degrees of carboxylation.
[0021] FIG. 2C shows the dependence of the gelation temperature of
agarose hydrogels as a function of the degree of carboxylation;
[0022] FIG. 2D shows the CD spectrum of a 0.15% wt/vol solution of
93% carboxyl modified agarose (93-CA) below the gelation
temperature (5.degree. C.) and above the gelation temperature
(90.degree. C.)
[0023] FIG. 3A shows the comparison of the rheological behaviour of
natural agarose and 60% carboxylated agarose (60 CA) through
comparison of shear modulus (G') and loss modulus (G'');
[0024] FIG. 3 B shows the shear modulus as a function of the degree
of carboxylation at various hydrogel concentrations;
[0025] FIG. 4A shows the organization of human umbilical vein
endothelial cells (HUVECs) into 2D lumens (L) in four types: oval
structures composed of a single cell, type I; elliptical structures
composed of two cells, type II; circular structures composed of two
to four cells, type III; and circular structures composed of more
than four cells, type IV. (Scale bar: 10 .mu.m.) The color-coded
bars at the bottom span morphologies typically observed under the
various conditions.
[0026] FIG. 4B shows a scatter plot of diameter and cell numbers
associated with lumens.
[0027] FIG. 4C shows large-scale organization of HUVECs.
[0028] FIGS. 4D-F show the apical-basal polarization of HUVECs in
CA60 gels.
[0029] FIG. 4G shows the mRNA expression level of key
provasculogenic markers in HUVECs. The expression of (from left to
right for each of the four samples) PODXL, LAM5, CCM1, NID2 and
COLIV in HUVECs in CA60 gels (Scale bar: 10 .mu.m).
[0030] It was an object of the present invention to provide
hydrogels, the physicochemical properties of which may be tuned
over a wide range of properties. These hydrogels preferably should
be suitable for use in therapeutic angiogenesis as described
above.
[0031] This object has been achieved by injectable hydrogels
comprising polysaccharides based on disaccharides the backbones of
which form an .alpha.-helix structure and in which in at least 10%
of the disaccharide units of the primary hydroxyl groups are
oxidized.
[0032] The term hydrogel, as used herein, is intended to denote a
water insoluble network of polymer chains in which water is the
dispersion medium. Hydrogels possess a degree of flexibility
similar to natural tissues.
[0033] Hydrogels are three-dimensional networks composed of
hydrophilic polymers crosslinked either through covalent bonds or
held together via physical intramolecular and/or intermolecular
attractions.
[0034] Hydrogels differ from normal gels in a number of properties.
Whereas gels are semi-solid materials made of hydrophilic polymers
comprising small amounts of solids dispersed in relatively large
amounts of liquid, hydrogels are also made up of hydrophilic
polymer chains, but these chains are crosslinked. This enables
hydrogels to swell while retaining their three dimensional
structure without dissolving. Thus, the principle feature of
hydrogels differentiating them from gels is their inherent
crosslinking.
[0035] The injectable hydrogels in accordance with the present
invention comprise polysaccharides based on disaccharides.
[0036] In a preferred embodiment the polysaccharide is derived from
agarose. Agar, a structural polysaccharide of the cell walls of a
variety of red seaweed, consists of two groups of polysaccharides,
namely agarose and agaropectin. Agarose is a neutral, linear
polysaccharide with no branching and has a backbone consisting of
1,3-linked .beta.-D-galactose-(1-4)-.alpha.-L-3,6 anhydrogalactose
repeating units. This dimeric repeating unit, called agarobiose
differs from a similar dimeric repeating unit called carrabiose
which is derived from carrageenan in that it contains
3,6-anhydrogalactose in the L-form and does not contain sulfate
groups.
[0037] Other polysaccharides which may be mentioned here are
hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin
sulfate, alginate, chitosan, pullulan and .kappa.-carrageenan.
[0038] Preferred examples of polysaccharides the backbones of which
form an .alpha.-helix structure are agarose and
.epsilon.-carrageenan.
[0039] The degree of oxidation of the primary hydroxyl groups may
vary over a wide range and is at least 10%, preferably at least
11%, more preferably at least 20% and most preferably 35% or more.
In some cases degrees of oxidation of from 50% to 95%, preferably
of from 55 to 93% have shown to be advantageous. While it is in
principle possible to completely oxidize the primary hydroxyl
groups, degrees of modification of at maximum 99%, preferably at
maximum 95% and even more preferably at max. 93% are preferred.
[0040] In certain cases oxidation of from 20 to 70%, preferably of
from 25 to 60% has proved to be advantageous.
[0041] The percentages for the degree of modification are in per
cent of the number of the respective groups in the
polysaccharide.
[0042] Preferably the primary hydroxyl groups are oxidized into
carboxylic acid groups.
[0043] The oxidation of primary hydroxyl groups in saccharide units
can be effected in a variety of ways which are known to the skilled
person, who will select the appropriate process based on his or her
professional experience and suitable for the specific needs of the
individual application.
[0044] Just by way of example, the oxidation with the well known
oxidizing agent TEMPO ((2,2,6,6-Tetramethyl-piperidin-1-yl)oxyl),
reactivated with NaOCl and catalyzed by potassium bromide may be
mentioned here. Sodium hydroxide may be added during the reaction
to maintain the optimum pH and to compensate the acidification of
the solution due to the formation of the carboxylic acid groups.
NaOH not only stabilizes the pH but also provides a quantitative
measurement of the degree of oxidation as it compensates the
carboxylic acid groups formed.
[0045] A possible side reaction, the conversion of the carboxylic
acid group formed into an aldehyde group can be compensated by the
addition of a reducing agent such as sodium borohydride
(NaBH.sub.4) which reduces the aldehyde formed back to the primary
alcohol which can then be oxidized into the carboxyl group
again.
[0046] The formation of carboxylic acid along the polysaccharide
backbone can be monitored by the amount of NaOH during the reaction
and afterwards by the quantitative analysis using FTIR
spectrometry.
[0047] In order to determine as precisely as possible the
percentage of the oxidized primary alcohol groups it is possible
either to perform the oxidation reaction in a controlled manner or
alternatively the polysaccharide is oxidized completely so that
about 100% of the primary alcohol groups are oxidized. Such
completely oxidized polysaccharide can be blended with unmodified
polysaccharide which may either be the same polysaccharide or
another polysaccharide. Thus, the chemical modification can be
precisely controlled during the reaction or by controlling the
blending with another polysaccharide or the same unmodified
polysaccharide.
[0048] In accordance with a preferred embodiment of the present
invention, the polysaccharide in the hydrogel is covalently
modified with cell adhesion motifs such as the integrin binding
sequence arginine-glycine-aspartic acid (RGD, which may occur in
various alternatives, e.g. cyclic RGD, or variants), or with
peptide sequences like YIGSR, IKVAV. MNYYSNS or PHSRN to name just
a few examples.
[0049] In accordance with another preferred embodiment, the
hydrogels in accordance with the present invention contain soluble
signals such as e.g. vascular endothelial growth factor (VEGF),
phorbol 12 myristate acetate (PMA), fibroblast growth factors
(FGF), insulin growth factors (IGF), transforming growth factor
beta-1(TGF-.beta.) or platelet derived growth factor (PDGF).
[0050] The hydrogels in accordance with the present invention in
accordance with another embodiment comprise components of the
extracellular matrix (ECM) e.g. basement membrane proteins (BMP)
such as collagen type 4 (Col4), laminins (LAM) or entactin (also
known as nidogen) or mixtures thereof.
[0051] The BMPs can be introduced into the hydrogel using e.g.
Matrigel, a gelatinous protein mixture commercially available from
various sources.
[0052] BMP mimicking peptide sequences or BMP's extracted from
other mammalian tissue than Matrigel may also be mentioned. The
skilled person will select the appropriate system based on the
individual needs of the specific application.
[0053] Polysaccharides whose backbones are organized into a-helices
such as agarose and .kappa.-carrageenan can undergo thermally
reversible gelation from aqueous solutions. The key step in their
physical gelation is the aggregation of the double-stranded
.alpha.-helices (24, 25). It has been reported that oxidation of
the D-galactose primary alcohol residue in agarose results in
weaker gels (26).
[0054] Introduction of charged moieties such as carboxylic acid
groups alters helical interactions and thus the gelation behaviour.
Carboxylation of the primary hydroxyl group is an example for this
effect.
[0055] The formation of double-stranded helices in proteins and
oligonucleotides is driven by the ability of the macromolecular
chains to form weak interactions through H bonds. It is therefore
reasonable to assume that the potential for the formation of such
interactions would be a function of the distance between the H
atoms and the electronegative oxygen (alcohol or carboxylic acid).
Carboxylation promotes separation of the polymer chains, which
diminishes the likelihood of H-bonding interactions. The analysis
of the frequency of H bonds on a per frame basis shows that
carboxylation indeed decreases the propensity of at least one
H-bond formation by over 75% compared with unmodified agarose.
These observations taken in sum suggest the possibility that the
introduction of a charged carboxylic acid group can potentially
alter the associative behavior of the agarose helices.
[0056] As has been found in the course of the present invention,
carboxylation promotes a .beta.-sheet secondary structure. One
potential outcome of introducing charges along a polymer backbone
is a transition of the polymer chains from a coiled morphology to a
more extended morphology due to increased electrostatic repulsion
between the chains (27).
[0057] In the following the results of various investigations are
described showing the influence of a partial oxidation of the
primary hydroxyl groups with agarose as preferred polysaccharide.
The results apply in an equal manner to other polysaccharides which
have backbones forming an .alpha.-helix structure.
[0058] The analytical methods described below were used as
follows:
Circular Dichroism
[0059] Circular dichroism spectra were obtained using a Jasco
spectropolarimeter
[0060] J-810 equipped with a Peltier temperature cell Jasco
PFD-425S. Solution of 0.15% w/v of agarose was made in Milli-Q
water at 90.degree. C. for 15 min then the solution was cooled down
at 5.degree. C. in the CD chamber for 30 min prior to measurement.
Each spectrum has been recorded three times and summed together.
Each spectrum for a given modification is a mean of three different
syntheses.
Zeta Potential
[0061] Zeta potential has been measured on a Beckman Coulter Delsa
Nano C particle analyzer. The same solutions have been used as for
the light scattering experiment. Measurements have been made in a
flow cell that has been aligned with the laser prior to every
measurement. Each measurement has been made three times and an
average has been calculated, each spectrum for a given modification
is a mean of three different syntheses.
ESEM
[0062] SEM pictures were obtain with a ref agarose gels of 2% w/v
were prepared and 2 ml of this solution was frozen dried for 24
hours under 0.1 mbar vacuum in a 5 ml glass vial. The sample has
then been vertically cut and the inside of the sample has been
imaged at different magnification. Images shown here are
representative of different areas of a given sample at different
magnification, which have been reproduced with three different gels
prepared from different batches.
AFM
[0063] AFM pictures were obtained with a Veeco Dimension 2100.
Samples were prepared on a 3 mm microscopic glass holder that had
been passivated. The glass slide was washed with 0.1 M NaOH and
dried in an oven. The dry slides were then passivated with few
drops of dichloromethylsilane. Two slides were sandwiched together
to have a uniform passivation. After 10 min the slides were washed
with water and the excess of dichloromethylsilane was washed with
soap and the slides were dried. Slides side was prepared in a
hydrophobic way. Agarose samples were prepared as 2% w/v gels and
25 .mu.l of the solution was poured onto an unmodified glass slide,
a dichloromethylsilane passivated slide was then adjusted on top of
the solution. Slides of 0.5 mm were put as spacer between the
hydrophobic and the normal glass slide, the whole montage was then
allowed to gel for 30 min at 4.degree. C. The upper slide
(hydrophobic) was removed thereafter and a thin layer of agarose
gel was obtained. This gel was then allowed to stabilize at room
temperature for 30 min before measurement in order to avoid any
shrinkage or dilatation of the gel during the measurement.
Molecular Dynamics (MD)
[0064] MD simulations have been done using the Desmond package of
the Maestro version 8.5 from Schrodinger. Initial conformation has
been obtained from the x-ray structure of the agarose that has been
downloaded from the PDB library. Modified agarose has been drawn
from the PDB file directly inside the Maestro software. Implicit
water model has been build using the Desmond tool, resulting in a
10 .ANG. square box build by following the TIP3 solution model. The
simulations have been run in the model NPV at 300.degree. K. at
atmospheric pressure for 15 ns. Analysis of the results was done
using the VMD software and the tools available in the standard
package.
[0065] A technique commonly used to study secondary structure in
biological molecules is circular dichroism (CD) (28). CD is very
sensitive toward changes in the coupling of transition dipole
moments, which serves as a probe for secondary structure, e.g.,
.alpha.-helices or .beta.-sheets in proteins (28). In unmodified
agarose the CD arises from coupling of C--O--C ether chromophores,
leading to positive residual ellipticity with a maximum at 183 nm
for .alpha.-helices (FIG. 2A) (29)). This ellipticity can be
directly attributed to the .alpha.-helices, as it is absent in
oligomeric agarose obtained from acid-catalyzed hydrolysis, which
is incapable of organizing into an .alpha.-helix.
[0066] The changes to the agarose secondary structure upon
carboxylation (to obtain carboxylated agarose, which will be
referred to hereinafter as CA), can be seen by using 93%
carboxylated agarose (93-CA) as a model system. Like unmodified
agarose (natural agarose, referred to hereinafter as NA), 93-CA
exhibits strong positive ellipticity; however, in comparison with
NA the maximum is even stronger and red-shifted (191 nm) (FIG. 1A)
and, additionally, the red shift is accompanied by the emergence of
a new peak at 203 nm. The new ellipticity at 203 nm can be
attributed to the carboxylation of the backbone, as its maximum
increases exponentially with carboxylation (FIG. 1B). The change in
molar absorptivity and shift to a lower energy excitation
wavelength of the primary ellipticity may also be due to
chromophore contributions of the introduced carboxyl group to the
network of dipolar couplings in the a-helices in 93-CA. It
therefore appears that the modification of the NA backbone promotes
a reorganization of the chains leading to a new secondary structure
in CA in addition to the native .alpha.-helices. In protein CD
spectra positive ellipticity around 217 nm indicates .beta.-sheets
(28). Likewise, the secondary structure-related ellipticity at 203
nm in the CD spectrum of 93-CA may be attributed to a
.beta.-sheet-like conformation of the polysaccharide chains.
Further evidence for the molecular reorganization leading to a new
secondary structure can be obtained by analyzing the molecular
dynamics (MD) simulation data. In proteins, the occurrence of
helical or .beta.-sheet motifs can be determined using the
empirical Ramachandran plot (30). Extending this approach to
polysaccharides (31), the empirical distributions of the dihedral
angles .phi. and .psi. of the glycosidic backbone were plotted
(FIG. 1C). As expected, in the case of NA the Ramachandran plot
reveals the predominance of helical conformation. However, in
contrast the coordinates of the CA dihedrals are mainly located in
the .beta.-sheet region consistent with the CD spectra, and this
implies a dramatic reorganization of the polysaccharide backbone
upon carboxylation. Such an .alpha.-helix to .beta.-sheet
transformation, although reported in proteins, is highly restricted
and has not been observed before in polysaccharides.
[0067] To ascertain the impact of the .beta.-sheet structure on the
organization of agarose molecules, tapping mode atomic force
microscopy (AFM) (32) was used to visualize NA and carboxylated
agarose (CA) molecules (FIG. 2A). It is clear that the NA strands
are organized as helical structures (FIG. 2A, Inset), appearing
like "a string of pearls"(FIG. 2A, Left). At 28% carboxylation
(28-CA), the helical organization appears slightly disrupted and
this is consistent with the CD data for 28-CA, where only a small
shoulder associated with the ellipticity at 203 nm is observed.
However, increasing carboxylation (60%, 60-CA; and 93%, 93-CA)
results in the complete reorganization of fibers into disk-shaped
structures that appear to have some residual helical motifs. Fibers
of soluble amyloid-.beta. (A.beta.) peptide fibrils that possess
mixed .beta.-sheet structures, also form circular globules, like
those observed in the 60-CA and the 93-CA (33, 34). Because the
molecular mass of agarose after oxidation [M.sub.n, 88-94 kDa;
polydispersity index (PDI), 2.14-2.23] is virtually identical to
that of NA (M.sub.n, 95 kDa; PDI, 2.99-3.12), its contribution to
the observed structural changes can be ruled out. The visual
evidence is consistent with the CD data and the Ramachandran plot
predictions, and is proof for the presence of a unique secondary
structure in CA.
[0068] Without being bound to any theory, one could postulate a
prominent role for reduced H bonding and increased electrostatic
repulsion between chains upon carboxylation, which, in sum, may
promote more hydrophobic interactions leading to hitherto unknown
interactions between agarose molecules.
[0069] In proteins, changes to secondary structure can alter
protein folding (tertiary structure) in a manner that favors
aggregation. In fact, soluble A.beta. has a disordered structure;
however, aggregates of A.beta. have a significant amount of
.beta.-sheet structure (35). If the paradigm for structure
evolution is conserved between polysaccharides and proteins, then
one might expect that the switch from a-helix to .beta.-sheet could
also impact the supramolecular assembly of the agarose molecules
and hence the microstructure of the gel. To determine whether this
indeed occurs and to what extent, the interior of freeze-dried 2%
wt/vol hydrogels was characterized using environmental scanning
electron microscopy (ESEM). Whereas the microstructure of the NA
gel was composed of tufts of disordered fibers, microstructures of
the CA gel bear no resemblance to NA and reveal an astonishing
transformation in the organization of agarose fibers with an
increasing degree of carboxylation (FIG. 2B). Even at a low degree
of carboxylation (28%), the fibers are organized into ridge-like
structures, composed of high-aspect ratio cells that appear to have
some periodicity. Increasing the carboxylation to 60% further
enhances this organization, wherein disk-shaped motifs appear to
fuse to one another in columnar strands organized into lamellae. At
93% carboxylation, the fiber organization appears to have undergone
a fundamental change, resulting in sheet-like structures composed
of highly oriented ribbons. Because 93-CA chains are organized into
disk-shaped structures, their assembly into sheets would require an
unraveling followed by lateral stacking. It has been shown that the
transformation of A.beta.42 disk-shaped oligomers into fibrils
involves organization of the peptide strands within these oligomers
into .beta.-sheets (36). The switching of the three-dimensional
(3D) structure of a polymer hydrogel from a random organization of
fibers to a lamellar structure has not been described before and
has been found in the course of this invention.
[0070] The gelation of NA involves association of .alpha.-helices
through H bonding mediated by the C6 primary hydroxyl group.
Because carboxylation modifies helical interactions, it also
imposes changes to the physicochemical characteristics of CA gels.
The gelation behavior of agarose shows a hysteresis in that the
melting temperature of the gel (T.sub.m, >80 .degree. C.) is
significantly higher than the gelation temperature (Tgel
.about.40.degree. C.) (23). This is expected, as the formation of
the gel requires H bonding, which is more likely to occur as the
entropy of the system is reduced. A key prediction of the MD
simulation is that CA chains have markedly diminished associative
tendencies, resulting in lower H-bond formation. One implication is
a decrease of the gelation temperature, as promotion of H bonding
requires lower kinetic energy. In fact, the complete carboxylation
of agarose results in the lowering of the Tgel to below 10.degree.
C. (i.e., .delta.=-30.degree. C.) over agarose, with intermediate
carboxylation yielding intermediate T.sub.gel (FIG. 2C).
[0071] The lower gelation temperature is advantageous for cell
encapsulation and tissue regeneration applications, as activation
of heat-shock proteins can be avoided (37).
[0072] Concrete proof for the direct involvement of the
.beta.-sheet in the gelation of the CA gels was obtained by
following the CD spectrum of 93-CA (fully carboxylated agarose) as
a function of temperature. The CD spectrum of the 93-CA gel above
its T.sub.m shows a complete abolishment of the ellipticity at 203
nm associated with the .beta.-sheet structure and additionally a
further red shift of the ellipticity at 199 nm in comparison with
the gel at 5.degree. C. (FIG. 2D). This is strong evidence that in
CA the new .beta.-sheet organization is responsible not only for
the physical cross-linking of the gel but also for the dominant
associative interaction between the polysaccharide chains.
[0073] Without being bound to any theory, the results obtained
would be in accordance with a mechanism for the gelation of CA
involving four steps of: (i) reorganization of the polymer backbone
due to disruption of helices, resulting in a-helix to .beta.-sheet
switch; (ii) followed by aggregation of polymer chains through
.beta.-sheet motifs; (iii) elongation of these aggregates into
high-aspect ratio structures; and (iv) the assembly of these
high-aspect ratio structures in higher lamellar sheets.
[0074] Cross-links are often described as knots or entanglements of
two and more chains. Because the gelation in both NA and CA can be
attributed to association of secondary structure of a specific
conformation, the cross-links can be imagined as assimilation of
these secondary structures into soft spheres, and its formation can
be linked to the growth of nano-particles through phase inversion.
These highly specific associative processes can manifest in dilute
solutions as aggregates. The size, polydispersity (PD), and zeta
potential (.zeta.) of aggregates that are spontaneously formed in
dilute solutions of NA and CA were determined using dynamic light
scattering. The average size of aggregates formed in NA solution
(0.15% wt/vol) was 1.09 .mu.m, with a PD of .about.0.6, suggesting
a rather heterogeneous associative process. In contrast, the
aggregates formed from CA solutions were almost half the size,
around 600 nm, and more narrowly dispersed (PD .about.0.3),
implying a higher homogeneity. In fact, the changes to the size of
the cross-links manifest themselves as a loss of turbidity in the
gels, which is concomitant with increased carboxylation.
[0075] As these aggregates represent the origins of physical
cross-linking, their charge characteristics influence their
crystallization into a gel network. Although aggregates of NA have
only a slightly negative .zeta. (-5 mV), .zeta. of the CA aggregate
becomes increasingly negative (maximum -27 mV). Rheology studies
reveal that increasing surface charge favors a more highly
organized structure (38) but a much looser association due to
electrostatic repulsion (39), thereby resulting in gels with a very
well-defined microstructure, but which are physically weaker. CA
gels have lower G' and G''in comparison with NA. A typical rheology
curve of storage (G') and loss (G'') modulus as a function of
angular frequency for NA and 60-CA is shown in FIG. 3A. The
reduction in G' is consistent with the predictions from the MD
simulations, as a lower tendency for H bonding coupled with an
increased charged density along the polysaccharide lowers friction
at the molecular level, thereby reducing the shear modulus of the
gel (39). More importantly, by varying the degree of carboxylation,
the G' of the hydrogels in accordance with the present invention
can be tailored independent of the polysaccharide concentration,
for example for a 2% wt/vol gel over four orders of magnitude (from
3.6.times.10.sup.4 Pa to 6 Pa), spanning the entire range of soft
tissues found in the mammalian anatomy (40), and over a slightly
reduced range of G' for a 4% wt/vol gel (FIG. 3B).
[0076] The reduction of the shear modulus G' of hydrogels based on
disaccharides by a process wherein the hydrogel is subjected to a
partial oxidation of the primary hydroxyl groups of the
disaccharides constitutes a further embodiment of the present
invention.
[0077] The origin of the changes to G' can be exclusively
attributed to the new secondary structure as both NA and CA have
identical molecular weights and PDI.
[0078] The ability to influence secondary structure of
polysaccharides via carboxylation is not limited to agarose but can
also be demonstrated in other polysaccharides. .kappa.-carrageenan,
a polysaccharide, like agarose, also organizes into helical
structures. Carboxylation of the primary alcohol at C6 position of
sulfated D-galactose in .kappa.-carrageenan results in changes to
the CD spectra that are identical to those in agarose. Upon
carboxylation, the negative residual ellipticity of
.kappa.-carrageenan .beta.-helices undergoes a red shift from 183
nm to 189 nm, which is again accompanied by a new residual
ellipticity with a maximum at 203 nm. The opposite sign of these
two spectral features clearly demonstrates the co-existence of two
secondary structural elements, .beta.-helix and .beta.-sheet, in a
single polysaccharide. Furthermore, AFM images reveal that
carboxylation of .kappa.-carrageenan promotes the transition of
polymer fibers from helical to disk-shaped assemblies as observed
in CA. Even more remarkable is that the switch from helices to
.beta.-sheets, like in the case of agarose, induces reorganization
of the microstructure of the freeze-dried gels from fibrous
(unmodified .kappa.-carrageenan, KC) to a high-ordered lamellar
structure (carboxylated .kappa.-carrageenan, CKC). The organization
of polymer fibers in carboxylated .kappa.-carrageenan is also
driven by the association of .beta.-sheets, like in CA, as can be
seen from a lower modulus for carboxylated .kappa.-carrageenan in
comparison with .kappa.-carrageenan. The G' for carboxylated
.kappa.-carrageenan is three orders of magnitude lower than that
for .kappa.-carrageenan.
[0079] This shows that carboxylation of primary hydroxyl groups in
hydrogels based on disaccharides provides a general approach for
altering the secondary structure of .alpha.-helical
polysaccharides.
[0080] CA60 Gels Promote Human Umbilical Vein Endothelial Cell
(HUVEC) Organization into Lumens. The organization of cells into
tissue-like structures involves a complex interplay between the
soluble signals and those originating from the ECM (matrix
stiffness, cell-ECM binding motifs, bound growth factors). Because
stiffness of a biomaterial has been shown to impact stem cell
lineage choices (1) and the metastasis of cancer cells (41), it can
be expected that the injectable CA gels with tunable mechanical and
structural properties in accordance with the present invention will
be highly desirable for cell delivery and as a clinically
translatable system for controlled tissue morphogenesis.
[0081] Vascularization is critical for the survival of cells and
necessary for the transport of signaling molecules to aid in
regeneration. Vasculogenesis, as it pertains to in vitro studies,
is the formation of lumens from dispersed endothelial cells (ECs),
and it differs from angiogenesis where endothelial structures form
from an already existing blood vessel or an EC monolayer (42). It
is well established that during vascular lumen morphogenesis, i.e.,
the formation of arteriole-like structures, cell-cell contacts and
mural (support) cells play a vital role. To identify the factors
that influence EC organization, several in vitro models have been
established, including collagen gel, fibrin gel, and matrigel
(43-46). These studies have revealed that arginine-glycine-aspartic
acid (RGD) integrin binding sequence and soluble signals such as
vascular endothelial growth factor (VEGF) and fibroblast growth
factor-2 (FGF-2) are essential and that the mechanical aspects of
the gel impact the formation of EC networks (43-46).
[0082] Nevertheless, the factors that impact the organization of
multiple ECs in freestanding tubular structures are not fully
understood. Because vasculogenesis primarily occurs during
embryonic development when ECM is immature, a screening of the
impact of the gel modulus and bound and soluble signals on human
umbilical vein endothelial cell (HUVEC) organization was performed
to evaluate the role of the immediate cellular environment in how
soluble and bound signals are perceived by ECs. To investigate this
premise further the organization of HUVECs in CA gels of two
moduli, 0.02 kPa (CA60) and 1 kPa (CA28) was studied by
systematically altering three parameters: basement membrane
proteins (.+-.0.01% wt/vol Matrigel), cell-binding motif (.+-.RGD),
and soluble signals [.+-.VEGF, FGF, and Phorbol 12-myristate
13-acetate (PMA)]. In comparison, HUVECs were also cultured in
fibrin gel and collagen gel supplemented with 0.01% Matrigel and
soluble signals and in Matrigel supplemented with soluble
signals.
[0083] The organization of HUVECs into lumens can be categorized
into four types as shown in FIG. 4A. In general, the organization
of HUVECs in fibrin and collagen gels involved one to two cells and
exhibited characteristics of type I and type II lumens (FIG. 4B).
However, in contrast, HUVECs in CA60 modified with RGD and
supplemented with basement membrane proteins and soluble signals
showed type III and type IV structures, with more than three HUVECs
participating in the formation of the lumens (FIG. 4B).
Interestingly, no such organization was observed in the series of
experiments for CA60 gels in the absence of RGD, basement membrane
proteins, and soluble factors. Analysis of the frequency and
structural characteristics (diameter and length) of the lumens
revealed significant differences. In general more lumens were
observed in CA60 gels in comparison with both fibrin and collagen
gels (FIG. 4B). Furthermore, despite higher cell numbers per lumen
the diameters of lumens formed in the CA60 gels were quite
homogeneous at around 50-100 .mu.m. However, more heterogeneity was
observed in collagen and fibrin gels (FIG. 4B). Another significant
observation was that HUVECs in CA60 gels could organize into
freestanding, hollow, tubular structures over 100 .mu.m in length,
resembling arterioles (FIG. 4C). In contrast, the average length of
such structures was about 50% smaller in collagen gels and an order
of magnitude lower in fibrin gels (FIG. 4C). This may be attributed
to the observed differences in the polarization potential of HUVECs
as discussed below.
[0084] Apical-basal polarization of ECs is a critical step in the
formation of stable blood vessels (47). Immunofluorescent staining
against human podocalyxin (PODXL) and type-4 collagen (COL4A1)
revealed apical and basal localization of PODXL and COL4A1,
respectively, in HUVECs in CA60 gels, suggesting that they had
undergone apical-basal polarization (FIG. 4D and E). In comparison,
HUVECs in fibrin and collagen gels did not stain for human PODXL
and COL4A1. This is consistent with the down-regulation at the mRNA
level of PODXL and NID2, both of which are necessary for lumen
expansion and maturation, in both collagen and fibrin gels in
comparison with CA60 (FIG. 4G). It is noteworthy that the lumens
appear to originate from a cluster of HUVECs that are already
polarized, i.e., show apical localization of PODXL. This is in
accordance with literature reports that vascular lumen
morphogenesis requires the polarization of an EC cluster (three to
five cells), which involves the recruitment of PODXL at the apical
surface, which then initiates lumen expansion (47). A factor that
might contribute to the formation of HUVEC clusters is the superior
proliferation of the HUVECs in the CA60-RGD-modified gel, which is
twofold greater than under expansion conditions on tissue culture
plastic. Interestingly, the incorporation of basement membrane
proteins, i.e., Matrigel, in the series of experiments made, had no
effect on lumen length in fibrin gels and provided only a marginal
increase in collagen gels (FIG. 4C) and this was also consistent
with the lack of appreciable changes to the expression of key
vasculogenesis markers at the mRNA level. This implies that the
observed organization of HUVECs into lumens in CA60 gels cannot be
attributed solely to the presence of growth factors and basement
membrane proteins because HUVECs in Matrigel while staining
positive for PODXL and COL4 do not show apical-basal localization
and also do not organize into lumens. A noteworthy observation is
that HUVECs in the CA28 gels, although showing comparable
expression levels of the provasculogenic markers in comparison with
HUVECs in CA60 gels at the mRNA level, however, remain dispersed
and fail to organize, thus suggesting a role for biophysical
variables.
[0085] Several mechanisms might contribute to the provasculogenic
characteristics of the low-modulus CA gels. In biological gels such
as fibrin and collagen, proteolytic degradation of the matrix by
membrane-type matrix metalloproteinases is necessary and critical,
as it paves the way for the migration and organization of the ECs
(43). CA without additional factors or components cannot undergo
proteolytic degradation, but could be slowly degraded through
hydrolytic dissolution and possibly other as yet not completely
understood mechanisms. At any rate, these are slow processes that
take place at a much longer time-scale (weeks) than endothelial
cell organization (1-2 days). Therefore, the role of matrix
degradation in the organization of HUVECs in CA gels can be ruled
out. However, it is possible that the stiffness and the chemistry
of the gel can modulate the organization and affinity of ECM
components and soluble signals. Because the stiffness of fibrin and
collagen gels is similar to that of CA60, this suggests that the
origin of the provasculogenic nature of CA60 may lie in its unique
physicochemical properties (backbone charge and secondary
structure) and not solely in its stiffness. Another aspect worth
considering is the modulation of HUVEC function by the CA gel
through a mechanobiology paradigm. This would require mechanical
coupling between the gel matrix and the cell through the RGD motif
and a unique role for this motif in HUVEC function. It has long
been recognized that RGD signaling is important for EC survival and
proliferation (48). Furthermore, it is well known that
.beta.1-integrin signaling is important in arterial tubulogenesis
and .beta.1 integrins have a binding site for RGD (49). Therefore,
it is conceivable that .beta.1 integrins on the HUVECs (50)
mechanically couple to the gel through the RGD ligand and thereby
sense the mechanical environment provided by the gel.
Interestingly, among 32 conditions screened, HUVEC organization
into lumens occurs only in CA60 gels modified with RGD. On the
basis of these findings, it can be concluded that CA gels are well
suited for understanding and leveraging the role of mechanobiology
in tissue morphogenesis and provide a potential translational
platform for regenerative therapies.
[0086] In this invention, injectable gels for Therapeutic
Angiogenesis are disclosed. In particular gels comprising of
carboxylated agarose of various degrees of carboxylation (CAXX,
where XX denotes the degree of carboxylation from 10-100),
optionally covalently modified with cell adhesion motifs such as
the integrin binding sequence arginine-glycine-aspartic acid (RGD)
and additionally containing VEGF, phorbol myristate acetate, and
basement membrane proteins laminin, collagen type IV and entactin
are disclosed. These gel formulations can induce morphologically
accurate and physiological functional blood vessels with
appropriate branching structures in vivo.
[0087] The following Examples show the hydrogels in accordance with
the invention and their use.
EXAMPLES
[0088] Modified agarose was obtained as follows:
[0089] Agarose type I has been obtained from Calbiochem. TEMPO
(((2,2,6,6-Tetramethylpiperidin-1-yl)oxyl), NaOCl, NaBH4, NaBr, EDC
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)), MES buffer
(2-(N-morpholino)-ethanesulfonic acid) have been obtained from
Sigma Aldrich and used as received. Solution of 0.5 M NaOH have
been freshly made every three months as well as solution of 5 M
HCl. Peptide GGGGRGDSP has been obtained from Peptide
International. Ethanol technical grade was used without any further
purification. De-ionized water was used for non-sterile
synthesis.
[0090] Agarose was modified under sterile conditions: All the
chemicals were dissolved in autoclaved water and filtered with a
0.2 .mu.m filter. All the glassware was autoclaved and the reaction
was conducted under a laminar flow. Agarose (1 g) was autoclaved in
MilliQ water. Autoclaved agarose was poured into a 3-necked round
bottom flask. A mechanical stirrer was adapted to one of the necks.
A pH-meter was adapted on the round bottom flask. The reactor was
then cooled down to 0-5.degree. C. and vigorously stirred. TEMPO
(0.160 mmol, 20.6 mg) was added, NaBr (0.9 mmol, 0.1 g) and NaOCl
(2.5 ml, 15% solution) was as well poured inside the reactor. The
solution was adjusted to pH=10.8 with HCI and NaOH. The pH was
maintained at 10.8 by adding NaOH. At the end of the reaction
NaBH.sub.4 (0.1 g) was added and pH=8 was reached. The solution was
stirred for 1 hour and NaCl (0.2 mol, 12 g) and ethanol (500 ml)
was added. The agarose was precipitated and extracted in a funnel.
The two layers were then filtered on a frit glass. The agarose was
then dialyzed in Spectra Pore 4, MWCO=12-14000 for 2 days and the
water was changed two times. Prior to dialysis the membranes were
left overnight in a 70% ethanol solution, 2 hours before use they
were rinsed in autoclaved water. Finally the product was put on a
Christ LD 2-8 LD plus at 0.1 mbar for the main drying and at 0.001
mbar during the desorption phase. Samples were put in round bottle
flask and frozen in liquid nitrogen bath on a rotary evaporator
modified for this purpose. Thin layers of frozen solution were
obtained on the flask wall reducing the lyophilization time.
[0091] Five different hydrogels were prepared as follows: [0092]
NA--Hydrogel of unmodified Natural Agarose [0093] CA60--soft
hydrogel, (no RGD) [0094] CA28--hard hydrogel (no RGD) [0095]
28RGD--CA28 covalently modified with RGD [0096] 6ORGD--CA60
covalently modified with RGD
[0097] NA stands for unmodified agarose, CA28 for agarose in which
28% of the primary hydroxyl groups are converted to carboxyl groups
and CA60 for an agarose where 60% of the primary hydroxyl groups
have been converted into carboxyl groups.
[0098] Growth Factors (GF) were introduced into the hydrogels using
Matrigel (Mat), obtaining seven hydrogel formulations as
follows:
[0099] 1 60RGD+Mat+GF
[0100] 2 28RGD+Mat+GF
[0101] 3 NA--unmodified control
[0102] 4 6ORGD+Mat
[0103] 5 28RGD+Mat
[0104] 6 CA60+Mat+GF
[0105] 7 CA28+Mat+GF
[0106] The different gels were preloaded inside insulin syringes
(BD Bioscience) and were kept on ice until injection.
In vivo Intramuscular Injection
[0107] Mice were anaesthetized before injection. 50 .mu.L of cold
phosphate buffer solution (PBS) were injected in the Gastrocnemius
(two injections per leg, i.e. 4 in total per animal). The cold PBS
injection was used to cool down the injection site before the
injection of the gel. Directly thereafter 50 .mu.L of the gel
formulations formulation was injected at the same injection point
that was marked with a pen. Only one condition was injected per
leg. The animals were left 2 weeks under normal diet and then
sacrificed.
Tissue Histology
[0108] Mice were anesthetized and tissues were fixed by vascular
perfusion with 1% paraformaldehyde in PBS pH 7.4. Gastrocnemius
muscles were harvested, embedded in OCT compound (CellPath,
Newtown, Powys, UK), frozen in freezing isopentane, and
cryosectioned. Sections of 25 .mu.m in thickness were stained with
the following primary antibodies and dilutions: rat monoclonal
anti-mouse CD31 (clone MEC 13.3, BD Biosciences, Basel,
Switzerland) at 1:100; mouse monoclonal anti-mouse .alpha.-SMA
(clone 1A4, MP Biomedicals, Basel, Switzerland) at 1:400; rabbit
polyclonal anti-NG2 (Chemicon International, Hampshire, UK) at
1:200. Fluorescently labeled secondary antibodies (Invitrogen,
Basel, Switzerland) were used at 1:200.
Intravascular Lectin Staining
[0109] Physiological perfusion of induced vessels was assessed by
intravascular staining with a fluorescently labeled Lycopersicum
esculentum (tomato) lectin or biotinylated Lycopersicon esculentum
lectin (Vector Laboratories, Burlingame, Calif., USA) that binds
the luminal surface of blood vessels. Biotinylated Lycopersicon
esculentum lectin was detected with fluorescently labeled
streptavidin (eBioscience, Vienna, Austria). Briefly, mice were
anesthetized and lectin was injected intravenously (50 .mu.l of a 2
mg/ml lectin solution per mouse) and allowed to circulate for 4 min
before vascular perfusion of 1% PFA in PBS pH 7.4 for 3 min under
120 mm/Hg of pressure.
Vessel Quantifications
[0110] Vessel diameters and vessel length density were measured in
muscle frozen sections after staining for CD31 (marker for
endothelial cells), NG2 (marker for pericyite cells) and SMA
(marker for smooth muscle cells). Briefly, vessel diameters were
measured by overlaying captured microscopic images with a square
grid. Squares were selected randomly and the diameter of each
vessel, if present, in the defined square was measured (in .mu.m).
At least 100 diameters were randomly quantified for each
experimental condition. Vessel length density was measured in at
least 15 representative fields per muscle tracing the total length
of vessels in each field and dividing it by the area of the field,
which was kept constant for all measurements and all experimental
conditions (mm of vessel length/mm.sup.2 of surface area). Vascular
segment length was defined as the average length (in .mu.m) of the
linear vessel segments comprised between two branch points. It was
also measured in the same analyzed microscopy fields by counting
the number of branching points in the vascular network (n) and
dividing the total vessel length by n+1. All analyses were
performed using the Cell P imaging software (Olympus, Volketswil,
Switzerland).
Statistics
[0111] The significance of differences was assessed using analysis
of variance (ANOVA) followed by the Sidak test for multiple
comparisons (GraphPad Prism 6). p<0.05 was considered
statistically significant.
[0112] The vessel growth induced by different compositions 2 weeks
after implantation and the corresponding quantifications of
vascular parameters were investigated. As expected, the unmodified
Agar condition (NA) also contained vascular structures, since it is
based on a fully biocompatible material. The CA28 and, more
extensively, the CA60 modifications alone caused a loss of vascular
ingrowth despite the combination with matrigel and GF, but this was
restored to various extents by the addition of the RGD sequence.
Analysis of vessel diameter showed a homogeneous distribution in
the size range of normal capillaries for all conditions, without
enlarged aberrant structures.
[0113] It is interesting to note that [0114] a) the presence of
matrigel did not improve the amount of vascular ingrowth; [0115] b)
the presence of matrigel even caused the formation of vascular
networks that are less branched, as evidenced by a longer average
segment length, and therefore have a less beneficial connectivity.
This is of some importance, because proper metabolic function of
newly induced vasculature requires an orderly branching so as to
achieve an efficient distribution of blood perfusion within the
tissue that should receive the nutrients and exchange waste
products; [0116] c) efficient vascular ingrowth occured even in the
absence of growth factors, i.e. it is not necessary to include
growth factor in the compositions; and [0117] d) the 28RGD and
60RGD conditions without either matrigel or GF display the shortest
segment length, i.e. the highest branching and connectivity of
induced vascular networks.
[0118] The 28RGD and 60RGD conditions, without addition of either
matrigel or GF, displayed the best examples of morphologically
ideal vascular structures, with properly branched capillary
networks, mature and associated with NG2-positive and SMA-negative
pericytes.
[0119] Lectin perfusion experiments showed that already after 2
weeks practically all induced vessels were functionally connected
with the general circulation, without major differences in any
condition, suggesting that in all conditions a process of
angiogenesis (growth of new vessels from pre-existing ones) takes
place rather than vasculogenesis (de novo assembly of endothelial
structures from progenitor cells).
[0120] In order to be therapeutically useful, newly induced
vascular structures must be able to persist long-term. This
important step is defined as vascular stabilization and has been
shown to take place within the first 4 weeks after induction of new
angiogenesis (51-53). Therefore, the stabilization afforded by the
different compositions to newly induced vascular structures was
investigated 7 weeks after hydrogel implantation in vivo.
[0121] The unmodified agar and 28RGD hydrogel conditions displayed
low vessel length density (VLD, which indicates the amount of new
vessels induced; the higher the value, the more new vessels are
induced), with poorly branched (segment length; the lower the
segment length the more branched the vessels are) and immature
endothelial structures, scarcely associated with NG2+/SMA-
pericytes, indicating a significant regression of the capillaries
that were induced after 2 weeks. Presence of GF in the 28RGD
condition allowed a better stabilization of induced vessels, which
however remained poorly covered by pericytes and poorly branched
(segment length).
[0122] In contrast, the 6ORGD hydrogel displayed very dense and
highly branched capillary networks, which were also mature, i.e.
associated with NG2-positive and SMA-negative pericytes.
Quantification of vessel length density showed that this
composition ensured complete stabilization of induced angiogenesis,
with no vascular regression and even further network expansion
compared with the 2-week time-point.
[0123] Remarkably the addition of GF did not provide any clear
benefit in terms of either amount of new vessels or branching of
the induced networks compared to 60RGD hydrogel alone.
[0124] Vessel diameters did not show any statistical significance
among the different groups.
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